Targeting dysfunctional developmental signaling cascades as a tool for tailored therapy in embryonal tumors

Neuroblastoma and medulloblastoma are embryonal malignant tumors of the central and peripheral nervous systems, respectively. The overall survival for both diagnoses is 60-75% despite intense multimodal therapy. This warrants the search for novel therapeutic approaches. Hedgehog (HH) and Wingless (WNT) signaling pathways are important regulators of embryogenesis that have been associated with the development of cancer including medulloblastoma. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors. WNT signaling is divided into canonical and noncanonical signaling. Canonical signaling involves the key molecule β-catenin while noncanonical signaling includes the planar cell polarity pathway (PCP). PCP signaling including the Rac/Rho cascade are important for proper migration and differentiation of neural crest cells during neuritogenesis. In this thesis we have assessed the role of HH and WNT signaling in neuroblastoma and medulloblastoma, with the aim to gain insights and develop novel therapeutic approaches based on biological understanding of the diseases.

Key molecules within the HH signaling pathway are overexpressed in neuroblastoma. We investigated the effects of inhibiting HH signaling in neuroblastoma and found that inhibition of GLI was more effective in reducing neuroblastoma growth compared to inhibition of SMO located more upstream in the signaling pathway. The GLI inhibitor GANT61 effectively repressed neuroblastoma growth in vitro and in vivo, downregulated c-MYC, GLI1, MYCN and Cyclin D1 expression and augmented the cytotoxic effects when combined with clinically used chemotherapeutic drugs. These findings suggest that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma (Paper I). The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is frequently overexpressed in cancers and is coupled to chemoresistance. In a search for new inhibitors for MGMT we investigated cellular regulators of MGMT expression in multiple cancers including neuroblastoma and medulloblastoma. We found a significant correlation between WNT signaling and MGMT expression that was confirmed by bioinformatic analysis and studies of protein expression of MGMT and β-catenin. Pharmacological as well as genetic inhibition of WNT activity downregulated the MGMT expression and restored chemosensitivity of the DNA-alkylating drug temozolomide in vitro and in vivo. These results have possible therapeutic entailments for chemoresistant cancers, especially of brain tumors where temozolomide is commonly used in treatment (Paper II).

Investigating non-canonical WNT signaling in neuroblastoma revealed that overexpression of PCP core genes Prickle1 and Vangl2 led to suppression of neuroblastoma cell growth and reduced Wnt/β-catenin signaling. On the other hand, overexpression of Vangl2 in neural stem cells produced accumulation of active β-catenin and decreased differentiation, suggesting different roles of PCP proteins in tumorigenic cells compared to normal cells (Paper III). Furthermore, genetic analyses demonstrated that neuroblastoma tumors harbor frequent mutations of genes controlling neuritogenesis associated with the Rac/Rho signaling cascade. The majority of these mutations were described to result in inhibition of Rac or activation of Rho. Inhibition of ROCK, a key enzyme downstream of Rho, resulted in differentiation, inhibition of neuroblastoma cell growth and migration and degradation of MYCN protein. Small molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth both in a transgenic and in a xenograft model. This study proposes that manipulation of Rho signaling might offer new therapeutic alternatives for neuroblastoma (Paper IV).

Taken together, the work in this thesis demonstrates that the embryonal signaling pathways HH and WNT may offer new therapeutic targets for neuroblastoma and medulloblastoma.

List of scientific papers

I. Wickström M, Dyberg C, Shimokawa T, Milosevic J, Baryawno N, Fuskevåg OM, Larsson R, Kogner P, Zaphiropoulos PG, Johnsen JI. Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo. International Journal of Cancer. 2013;132(7):1516-24
https://doi.org/10.1002/ijc.27820

II. Wickström M, Dyberg C, Milosevic J, Einvik C, Calero R, Sveinbjörnsson B, Sandén E, Darabi A, Siesjö P, Kool M, Kogner P, Baryawno N, Johnsen JI. Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance. Nature Communications. 2015;6:8904.
https://doi.org/10.1038/ncomms9904

III. Dyberg C, Papachristou P, Haug BH, Lagercrantz H, Kogner P, Ringstedt T, Wickström M, Johnsen JI. Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma. BMC Cancer. 2016;16(1):259
https://doi.org/10.1186/s12885-016-2293-2

IV. Dyberg C, Fransson S, Sveinbjönsson B, Lännerholm-Palm J, Forsberg D, Herlenius E, Martinsson T, Kogner P, Johnsen JI , Wickström M. Rhoassociated kinase is a potential therapeutic target in neuroblastoma. [Manuscript]